Project Summary The mechanism of toxicity for the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds remains poorly understood, beyond activation of the aryl hydrocarbon receptor (AhR). TCDD induces xenobiotic metabolizing enzymes with subsequent increases in reactive oxygen species (ROS). Epidemiological and rodent studies have further implicated AhR activation in the development and progression of non-alcoholic fatty liver disease (NAFLD). Our preliminary data demonstrates that AhR ligands induce a novel antioxidant mechanism involving the expression of the pyruvate kinase M2 (PKM2) isoform. PKM2 expression causes metabolic reprogramming that redirects accumulating glycolytic intermediates to the pentose phosphate pathway (PPP) and serine biosynthesis to increase NADPH levels and glutathione production in of support cellular antioxidant responses. This proposal will establish a mechanistic link between AhR activation, metabolic reprogramming, antioxidant defenses, and progression of NAFLD pathologies in mouse and human models by (1) demonstrating AhR regulation of Pkm2 expression, (2) tracking the redirection of 13C-glucose and 13C-glutamine intermediates to the PPP and glutathione biosynthesis, and (3) investigating the antioxidant role of Pkm2 in the progression of hepatic steatosis to steatohepatitis with fibrosis, modulation of tumor surveillance immune cell populations, and dysbiosis of the gut microbiome. Collectively, these studies will demonstrate that AhR-mediated PKM2 induction is a novel cellular defense mechanism, and represents a major advancement in the elucidation of the hepatotoxicity of TCDD and related compounds, with a focus on the development and progression of NAFLD.